Abstract
An increased infection incidence of Candida albicans (most common human fungal pathogen) contributes to the need of further functional genetic studies and development of new antifungal drugs. We developed a method to create mutants of C. albicans using an antisense cDNA library to interfere with gene expression, followed by screening for hypersensitivity to Calcofluor White (CFW) and the antifungal drugs caspofungin and itraconazole. Mutants with these properties have with a high probability defects in cell-wall integrity. Fifty out of 200 transformant colonies analyzed (25 %) showed hypersensitivity to CFW compared with the parental strain C. albicans CAI-4. Most of those CFW-hypersensitive mutants further displayed the susceptibility to antifungal drugs itraconazole and caspofungin using microbroth dilution method M27-A and an agar-diffusion test. The mutants obtained through this procedure could provide a potential model for screening antifungal pro-drugs which show weak action when standard C. albicans strain is used and may also aid in further identifying genes involved in cell integrity. In addition, we describe the effect of varying several parameters in electroporation transformation, including treatment with lithium acetate, upon the efficiency of transformation in C. albicans.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Angiolella L., Micocci M.M., D’ALESSIO S., Girolamo A., Maras B., Cassone A.: Identification of major glucan-associated cell wall proteins of Candida albicans and their role in fluconazole resistance. Antimicrob.Agents Chemother.46, 1688–1694 (2002).
Balashov S.V., Park S., Perlin D.S.: Assessing resistance to the echinocandin antifungal drug caspofungin in Candida albicans by profiling mutations in FKS1. Antimicrob.Agents Chemother.50, 2058–2063 (2006).
Boorsma A., De Nobel H., Ter Riet B., Bargmann B., Brul S., Hellingwerf K.J., Klis F.M.: Characterization of the transcriptional response to cell wall stress in Saccharomyces cerevisiae. Yeast21, 413–427 (2004).
Chandrasekar P.H., Sobel J.O.: Micafungin: a new echinocandin. Clin.Infect.Dis.42, 1171–1178 (2006).
Damveld R.A., Vankuyk P.A., Arentshorst M., Klis F.M., Van Den Hondel C.A., Ram A.F.: Expression of agsA, one of five 1,3-α-d-glucan synthase-encoding genes in Aspergillus niger, is induced in response to cell wall stress. Fungal Genet.Biol.42, 165–177 (2005).
De Backer M.D., Maes D., Vandoninck S., Logghe M., Contreras R., Luyten W.H.: Transformation of Candida albicans by electroporation. Yeast15, 1609–1618 (1999).
De Backer M.D., Nelissen B., Logghe M., Viaene J., Loonen I., Vandoninck S., De Hoogt R., Dewaele S., Simons F.A., Verhasselt P., Vanhoof G., Contreras R., Luyten W.H.: An antisense-based functional genomics approach for identification of genes critical growth of Candida albicans. Nat.Biotechnol.19, 235–241 (2001).
Herreros E., Garcia-saez M.I., Nombela C., Sanchez M.: A reorganized Candida albicans DNA sequence promoting homologous non-integrative genetic transformation. Mol.Microbiol.6, 3567–3574 (1992).
Hill T.W., Loprete D.M., Momany M., Ha Y., Harsch L.M., Livesay J.A., Mirchandani A., Murdock J.J., Vaughan M.J., Watt. M.B.: Isolation of cell wall mutants in Aspergillus nidulans by screening for hypersensitivity to Calcofluor White. Mycologia98, 400–410 (2006).
Kapteyn J.C., Van Den Ende H., Klis F.M.: The contribution of cell wall proteins to the organization of the yeast cell wall. Biochim. Biophys.Acta1426, 373–383 (1999).
Lussier M., White A.M., Sheraton J., Di Paolo T., Treadwell J., Southard S.B., Horenstein C.I., Chen-weiner J., Ram A.F.J., Kapteyn J.C., Roemer T.W., Vo D.H., Bondoc D.C., Hall J., Zhong W.W., Sdicu A.M., Davies J., Klis F.M., Robbins P.W., Bussey H.: Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae. Genetics147, 435–450 (1997).
Maeda H., Ishida N.: Specificity of binding of hexopyranosyl polysaccharides with fluorescent brightener. J.Biochem.62, 276–278 (1967).
Marr K.A., Rustad T.R., Rex J.H., White T.C.: The trailing end point phenotype in antifungal susceptibility testing is pH dependent. Antimicrob.Agents Chemother.43, 1383–1386 (1999).
Mouyna I., Fontaine T., Vai M., Monod M., Fonzi W.A., Diaquin M., Popolo L., Hartland R.P., Latgé J.P.: Glycosylphosphatidylinositol-anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall. J.Biol.Chem.275, 14882–14889 (2000).
NCCLS (National Committee for Clinical Laboratory Standards): Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard M27-A. NCCLS, Wayne (PA, USA) 1997.
Noble S.M., Johnson A.D.: Genetics of Candida albicans, a diploid human fungal pathogen. Ann.Rev.Genet.41, 193–211 (2007).
Oliver S.: Redundancy reveals drugs in action. Nat.Genet.21, 245–246 (1999).
Onishi J., Meinz M., Thompson J., Curotto J., Dreikorn S., Rosenbach M., Douglas C., Abruzzo G., Flattery A., Kong L., Cabello A., Vicente F., Pelaez F., Diez M.T., Martin I., Bills G., Giacobbe R., Dombrowski A., Schwartz R., Morris S., Harris G., Tsipouras A., Wilson K., Kurtz M.B.: Discovery of novel antifungal (1,3)-β-d-glucan synthase inhibitors. Antimicrobial.Agents Chemother.44, 368–377 (2000).
Orłowski J., Machula K., Janik A., Zdebska E., Palamarczyk G.: Dissecting the role of dolichol in cell wall assembly in the yeast mutants impaired in early glycosylation reactions. Yeast24, 239–252 (2007).
Pardini G., De Groot P.W., Coste A.T., Karababa M., Klis F.M., De Koster C.G., Sanglard D.: The CRH family coding for cell wall glycosylphosphatidylinositol proteins with a predicted transglycosidase domain affects cell wall organization and virulence of Candida albicans. J.Biol.Chem.281, 40399–40411 (2006).
Pfaller M.A., Diekema D.J.: Epidemiology of invasive candidiasis: a persistent public health problem. Clin.Microbiol.Rev.20, 133–163 (2007).
Plaine A., Walker L., Da Costa G., Mora-montes H.M., Mckinnon A., Gow N.A., Gaillardin C., Munro C.A., Richard M.L.: Functional analysis of Candida albicans GPI-anchored proteins: roles in cell wall integrity and caspofungin sensitivity. Fungal Genet.Biol.45, 1404–1414 (2008).
Popolo L., Gilardelli D., Bonfante P., Vai M.: Increase in chitin as an essential response to defects in assembly of cell wall polymers in the ggp1δ mutant of Saccharomyces cerevisiae. J.Bacteriol.179, 463–469 (1997).
Ram A.F., Klis F.M.: Identification of fungal cell wall mutants using susceptibility assays based on Calcofluor white and Congo red. Nat.Protoc.1, 2253–2256 (2006).
Ram A.F., Kapteyn J.C., Montijn R.C., Caro L.H., Douwes J.E., Baginsky W., Mazur P., Van Den Ende H., Klis F.M.: Loss of the plasma membrane-bound protein Gas1p in Saccharomyces cerevisiae results in the release of β-1,3-glucan into the medium and induces a compensation mechanism to ensure cell wall integrity. J.Bacteriol.180, 1418–1424 (1998).
Raška M., Běláková J., Krupka M., Weigl E.: Candidiasis — do we need to fight or to tolerate the Candida fungus? Folia Microbiol.52, 297–312 (2007).
Richard M., Ibata-ombetta S., Dromer F., Bordon-pallier F., Jouault T., Gaillardin C.: Complete glycosylphosphatidylinositol anchors are required in Candida albicans for full morphogenesis, virulence and resistance to macrophages. Mol.Microbiol.44, 841–853 (2002).
Sanglard D., Ischer F., Monod M., Bille J.: Susceptibilities of Candida albicans multidrug transporter mutants to various antifungal agents and other metabolic inhibitors. Antimicrob.Agents Chemother.40, 2300–2305 (1996).
Thompson J.R., Register E., Curotto J., Kurtz M., Kelly R.: An improved protocol for the preparation of yeast cells for transformation by electroporation. Yeast14, 565–571 (1998).
Torosantucci A., Bromuro C., Chiani P., De Bernardis F., Berti F., Galli C., Norelli F., Bellucci C., Polonelli L., Costantino P., Rappuoli R., Cassone A.: A novel glyco-conjugate vaccine against fungal pathogens. J.Exp.Med.202, 597–606 (2005).
Vale-silva L.A., Buchta V., Valentová E.: Effect of subinhibitory concentration of some established and experimental antifungal compounds on the germ tube formation in Candida albicans. Folia Microbiol.52, 39–43 (2007).
Walther A., Wendland J.: An improved transformation protocol for the human fungal pathogen Candida albicans. Curr.Genet.42, 339–343 (2003).
Wang S., Luo Y., Yi X., Yu W., Xu Z., Ma X., He J., Liu Q.: A highly efficient and highly reliable protocol for transformation of Escherichia coli by electroporation. J.Rapid Meth.Autom.Microbiol.15, 253–258 (2007).
Author information
Authors and Affiliations
Corresponding authors
Additional information
Zhu-Mei He and Jing Chen contributed equally to this work.
Rights and permissions
About this article
Cite this article
He, ZM., Chen, J., Li, HZ. et al. Mutants of Candida albicans hypersensitive to calcofluor white display susceptibility to antifungal drugs. Folia Microbiol 55, 159–166 (2010). https://doi.org/10.1007/s12223-010-0024-7
Received:
Revised:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12223-010-0024-7